Difference between revisions of "Part:BBa K3350858"

Revision as of 06:52, 26 October 2020

yhaJ1st (DNA-binding transcriptional activator)
Several transcriptional regulatory protein families are involved in the metabolism of aromatic compounds in bacteria, and have difference in both their structures and in the activation mechanisms. The LysR-type transcriptional regulators (LTTRs) are characterized by an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain. According to Tropel and V an Der Meer, all LTTRs involved in aromatic compounds degradation pathways can act as transcriptional activators with the co-inducers, which are either degraded compounds or intermediates in the degradation pathways. The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF promoter (BBa_K1316002).[1]

Usage and Biology

The YhaJ protein, a member of the LysR type family, acts as a transcriptional regulator to activate the the yqjF promoter (BBa_K1316002).We generated the yqjF3rd (BBa_K3350862)promoter that show much reduced DNT detection threshold of 5 mg/L versus 25 mg/L of the wild-type yqjF promoter,and we overexpressed the yhaJ1st gene together with an yqjF3rd-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the yqjF promoter from 25 mg/L to 0.1 mg/L, which is a 250-fold increase of the sensitivity.

Characterization and Measurement

We had optimized the yqjF promoter to obtain its new version yqjF3rd (BBa_K3350862). Thus, when we overexpressed the yhaJ gene together with an yqjF3rd-containing reporter construct in the engineered bacteria, we could reduce the DNT detection threshold of the yqjF promoter from 25 mg/L to 0.25 mg/L, which is a 100-fold increase of the sensitivity.

Given the importance of the YhaJ transcription factor, we used random error prone PCR mutagenesis to mutated the yhaJ coding sequence. Based on the experimental results, we obtained an improved version of the transcription factor, yhaJ1st.

When we overexpressed the yhaJ1st, we observed the reduced DNT detection threshold of the yqjF promoter from 25 mg/L to 0.1mg/L, which is a 250-fold increase of the sensitivity.

References

[1] Palevsky N, Shemer B, Connolly JP, Belkin S. The Highly Conserved Escherichia coli Transcription Factor YhaJ Regulates Aromatic Compound Degradation. Front Microbiol. 2016 Sep 22;7:1490.

Sequence and Features